Technical Field
The present invention relates to a peptide nucleic acid of porcine reproductive and respiratory syndrome virus (PRRSV) and use thereof.
Related Art
Porcine reproductive and respiratory syndrome (PRRS) is a viral infectious disease caused by PRRS virus (PRRSV) and mainly characterized by swine reproductive disorder and Porcine Respiratory Disease Complex (PRDC). In 1987, PRRSV was initially found in the midwestern United States, and spread rapidly throughout the whole country, and then widespread around the world in a few years, causing a heavy economic loss to the swine industry. In 1992, the virus was officially termed as PRRSV on the International Conference of Virology. In 1996, PRRSV was initially isolated by Guo Baoqing et al from pigs with suspected PRRSV infection in China, thus confirming the existence of this virus in China. At present, PRRSV has become one of the vital pathogens causing infectious diseases to swine in China. The diseases are designated as category B infectious disease by the Office International Des Epizooties (OIC), and also category II infectious disease in China. Among them, high 2 pathogenic swine of blue ear disease is a class of infectious diseases.
The latency of PRRS is generally 3-24 days. Once present in the swine herd, PRRSV spreads rapidly, and the serum positive rate can generally be up to 85-95% in 2-3 months. The infected pigs have the symptoms of poor mental state, poor appetite or loss of appetite, elevated body temperature of 40.2-42.0° C., cough, and respiratory distress. After attack by the disease, the pigs at various ages show breathing difficulty, but the specific symptoms are not completely the same.
After being infected, the sows initially show anorexia, elevated temperature, shortness of breath, runny nose and other symptoms similar to the flu; a few (2%) show cyanosis of ends of the limbs, tail, nipple, vagina and ear tip, and cyanosis of the ear tip is mostly common; and very few sows may suffer from diarrhea. Finally, the paralyzed limbs and other symptoms may occur and usually last 1-3 weeks, and the sows may eventually died of failure. Sows in early pregnancy experience abortion, sows in middle pregnancy experience stillbirth, mummification, or give birth to weak or abnormal fetus, and sows in breast feeding fail to lactation post partum, causing the sucking pigs to starve to die.
After being infected, the boars show cough, sneezing, depression, inappetence, shortness of breath, movement disorders, decreased sexual desire, decreased semen quality, and less ejaculation.
After being infected, the growing finishing pigs and weaned piglets mainly show anorexia, lethargy, cough, and dyspnea, some pigs have swollen eyes, conjunctivitis and diarrhea, some weaned piglets have diarrhea, arthritis, red ears, and skin spots. Sick pigs often die of secondary infection with pleurisy, streptococcicosis, and asthma. If there is no secondary infection, the growing finishing pigs may be recovered.
After being infected, the suckling piglets have the symptoms of coarse hair, depression, dyspnea, asthma or ears cyanosis. Some have bleeding tendency, subcutaneous plaques, arthritis, sepsis and other symptoms, and the mortality rate is as high as 60%. The mortality of the piglets before weaning is increased, and the peak time usually lasts for 8-12 weeks. Once infected with the virus in the embryonic period, the newborns die at birth a few days after death, and the mortality rate is as high as 100%.
PRRSV is a member of the genus Arterivirus, which is an enveloped, non-segmented, single-stranded, positive-sense RNA virus. The virus is of spherical shape having a diameter of 50-65 nm, and has a nucleocapsid that is a symmetric icosohedron and has fine fibrous protrusions on surface thereof. The virus includes two serotypes, that is, the North American type, and the European type, and the strain isolated in China is the North American type. The genome has a full length of about 15 kb, and comprises 8 open reading frames (ORFs), in which adjacent reading frames are partially overlapped. An about 12 kb-long ORF1 is present at the 5′ terminus. ORF1 comprises ORF1a and ORF1b having 16 nucleotides overlapped, and accounts for 80% of the whole genome, thus giving rise to a non-structural protein (encoding RNA replicase and polymerase). The encoding region of ORF1a includes some hydrophobic regions, a putative serine protease region, and a cysteine-rich region. The leader sequence immediately adjacent to the initiation codon of ORF1a has two hairpin structures. The encoding initiation region of ORF1a has a core sequence of 5′-UAACCAU-3′, and is highly conserved. ORF1b comprises 4 domains: (1) a motif sequence of polymerase; (2) a zinc finger region rich in cysteine and histidine; (3) a motif sequence of helicase; and (4) a conserved region with unknown functions. A 5 non-coding region (NCR) of about 180-220 nucleotides in length is present upstream of ORF1, which is highly conserved and functions to initiate the translation of subgenomic mRNAs as a 5-terminal leader sequence. The gene expression pattern of PRRSV is expression by producing 6 subgenomic mRNAs that have a consensus leader sequence derived from 5 NCR of the viral genome, and share a common 3 nested structure. 6 encoding frames (ORF2-7) are present at the 3 terminus, which encode the viral structural proteins. The envelope proteins encoded by ORF2-ORF5 are GP2, GP3, GP4, and GP5 respectively, the membrane matrix protein encoded by ORF6 is the M protein, and the nucleocapsid protein encoded by ORF7 is the N protein. The termination codon of ORF7 is followed by a 3 non-coding region containing a Poly(A) tail, i.e. a conserved sequence of about 10 nucleotides in length, which is a region recognized and bound by an enzyme during viral replication, to initiate the synthesis of a negative stranded RNA. Among the structural proteins of PRRSV, GP5, M, and N are the primary structural proteins, and GP2a, GP3, GP4 and E are the secondary structural proteins, which are present at a low level in virions, and the function of which are less studied. It is confirmed by Welch et al that ORF2 and ORF4 deleted infectious molecular clones of PRRSV can be rescued in cell lines providing the correspondingly deleted proteins, to produce infectious virions. Whether GP3 is present in the virion is a matter in dispute. In LDV, GP3 is proved to be a non-structural, soluble glycoprotein that can be secreted from infected cells. In EAV, GP3 is proved to be assembled into virions in the form of structural protein. Both of the two cases may occur to PRRSV. The GP3 protein of European-type PRRSV LV isolate is proved to be present in the virion, and the GP3 protein of North American type isolate is proved to be present in the form of secretion (sGP3). The E protein is a newly identified small non-glycosylated hydrophobic envelope protein, which has a potential N-terminal N-myristoylation site and a tyrosine kinase II phosphorylase site, a central hydrophobic domain, and a hydrophilic C terminus rich in alkaline residues, and is present in all arteritis virions. The translation initiation codon of mutant E protein causes the infectious molecular clone of EAV to lose the infectivity.
A wide range of variations exist in the nucleotide sequence of PRRSV isolate. Substitution, insertion, deletion or gene recombination may occur to PRRSV at the nucleotide and amino acid levels. Studies show that variant blue-ear pig disease virus may cause “high fever” of swine, which is termed as “highly pathogenic blue ear disease of swine” for discrimination from ordinary blue ear disease. Compared with the blue-ear pig disease virus prevalent in China in 1996, the virulence and pathogenicity of the variant strain are significantly boosted. In 2006, in pig farms in some southern provinces of China, swine “high fever” syndrome broke out, in which the infected pigs mainly show the clinical characteristics such as high body temperature, high incidence and high mortality. Subsequently, it is confirmed through study that the main pathogen of the disease is PRRSV with deletion variation. The genetic variations of GP5 protein of PRRSV occurring from 1996 to 2006 in mainland of China are analyzed by Tong Guangzhi et al, and the results show that the PRRSV strains occurring from 1996 to 2006 in mainland of China are highly homogenous to strain VR22332 (representative strain of North American type virus) in terms of the amino acid sequence (85.5%-99.0%), and lowly homogenous to strain LV (representative strain of European type virus) (53.5%-57.0%). This suggests that the strains prevalent in China are all North American type, and can be classified into two subgroups that are far in inheritance relationship. All the strains of subgroup I have variations at the antibody binding site of the primary antigen neutralization epitope of the virus, and the subgroup II is highly conserved at this site. Although the genome of PRRSV has wide genetic variations, the M and N proteins are relatively conserved in all strains. The M protein gene is the most conserved, and the GP5 gene has the highest sequence difference.
ORF1 encodes the viral replicase. After processing, the oligomeric protein encoded by ORF1a forms 6 non-structural proteins (Nsp1α, Nsp1β, and Nsp2-Nsp5), where Nsp2 varies highly between the North American type and European type strains, and the homogeneity of amino acid sequence is merely 32%. The oligomeric protein encoded by ORF1b is about 1463 amino acids long, and cleaved by the protease encoded by ORF1a, to form 4 proteins, that is, RdRp, CP2, CP3, and CP4. GP2 is a glycoprotein encoded by ORF2, and has a molecular weight of 29-30 ku. The PRRSV GP2 of both gene types comprises 2 apparent hydrophobic peaks and 2 inferred N-glycosylation sites. GP3 is a glycoprotein encoded by ORF3, which has a molecular weight of 27-29 ku, is one of the proteins having the worst conservation among the strains of PRRSV, and has an inferred homogeneity of amino acid sequence of 54%-60% between the North American type and European type strains, with most of the variations occurring at the N terminus. GP4 is a glycosylated envelope protein encoded by ORF4, which has a molecular weight of 19-20 ku, comprises 4 glycosylation sites, and has a highly hydrophobic region at the N and C terminus. GP5 is a glycosylated envelope protein encoded by ORF5, which is also referred to as E protein, and has a molecular weight of about 22.4 ku; the GP5 proteins derived from the North American type and European type isolates comprise 200 and 201 amino acids respectively, have 6 antigenic determinants, and can induce an organism to produce specific neutralizing antibodies. The M protein is a membrane matrix protein encoded by ORF6 and has a molecular weight of 18-19 ku; and the amino acid sequences of the M protein between the North American type and European type strains are inferred to be the most conserved, comprise 173 and 174 amino acids, and have a homogeneity of 78-81%. The N protein is a nucleocapsid protein encoded by ORF7, which has a molecular weight of 14-15 ku, and is the smallest primary structural protein in PRRSV. The N protein of the North American type and European type PRRSV strains comprises 123 and 128 amino acids respectively, and the N protein of the European type strain has two more amino acid extensions at the N and C terminus than the North American type strain, which are STAPM and SQGAS respectively.
Antisense nucleic acid is a fragment of naturally occurring or artificially synthesized nucleotide sequence that is complementary to a sequence of a target gene (mRNA or DNA), and specifically binds to the viral target gene by base pairing to form a hybrid molecule, thus playing a role in the regulation of target gene expression at the level of replication, transcription, or translation, or in the induction of RNase H to recognize and cleave mRNA such that the function of mRNA is lost.
The antisense nucleic acid includes antisense RNA and antisense DNA, and is characterize by convenient synthesis, simple sequence design, easy modification, high selectivity, and high affinity. As a new anti-viral and anti-tumor agent, the antisense nucleic acid arouses a revolution in the field of pharmacology, that is, new reactions post drug-receptor binding are initiated by a new drug receptor mRNA through the new binding pattern to the receptor (Watson-Crick crossing), including: (1) degradation of the target RNA mediated by RNase H; and (2) inhibition on the DNA replication and transcription and post-transcriptional processing and translation, etc. It is believed that the antisense oligonucleotide (ODNs) therapy is more specific than the conventional drug therapies. Since the late 1970s, the antisense nucleic acid drugs have went out of the laboratory, and put into practical clinical use in the over three decades of years. The antisense therapy receives great attention especially after the first antisense nucleic acid drug Fomivirsen is approved by FDA.
The mechanism of action of antisense nucleic acids is that based on the principle of base pairing, it is involved in the regulation of relevant gene expression by binding to the target RNA through base pairing. The modes of action may include the following. (1) The anti-sense RNA is bound to the viral mRNA, to from a complementary duplex, thus blocking the binding of ribosome to viral mRNA, and inhibiting the translation of viral mRNA into proteins. (2) The anti-sense DNA can form a triple helix nucleic acid with the target gene, and regulate the transcription of a gene by acting on the transcript, enhancer and primer region controlling the gene transcription. (3) The binding of the antisense nucleic acid to the viral mRNA can prevent the transport of the mRNA to cytoplasm. (4) After the binding of the antisense nucleic acid to the viral mRNA, the mRNA are more easily recognized and degraded by the nuclease, thus greatly reducing the half life of mRNA. The four pathways of action may all be embodied as the inhibition or regulation for viral gene expression, and the regulation is highly specific.
The antisense nucleic acid recognizes the targeting gene based on the principle of base complementation and pairing. Theoretically, for example, the chromosome of animal cells has about several billions of pairs of bases. If the number of the 4 bases (A, G, C, and T) are substantially the same and distributed at random in the whole gene, then the antisense nucleic acid of greater than 17 bases is unlikely to hybridize to a non-target gene according to the principle of statistics. Therefore, the binding of the antisense nucleic acid molecule of greater than 17 bases to the target gene is unique, such that the antisense nucleic acid is highly specific.
Studies show that a copy of gene in the cell can produce 200-300 mRNAs, from which 100,000 biologically active protein molecules are translated. The conventional drugs mainly act on several sites on a domain of the protein molecule having biological functions. Actually, the protein structure is very complex and the spatial structure of active proteins in an organism is versatile. It is difficult to achieve a desirable effect by controlling the dynamics and overall functions of the target molecules via the limited several sites on which the conventional drugs act. Therefore, the limitation of the conventional drugs is obvious. Several dozens to hundreds of protein may be translated from the mRNA, and the target gene is directly regulated by the antisense nucleic acid at the mRNA level, which means that the efficacy of the conventional drugs is increased by several dozens to hundreds of times. It can be seen that the regulation by antisense nucleic acid is quite economic and reasonable.
Toxicological research shows that the antisense nucleic acid has an extremely low toxic in vivo. Although the antisense nucleic acid may remain in vivo for a long or short period of time, it is finally removed by degradation, through which the hazard caused by integration of an exogenous gene into the chromosome of a host in a transgenic therapy is avoided. Compared with the conventional drugs, the antisense nucleic acid drugs have the advantages of high specificity, high efficacy, and low toxic effect, and are useful in the inhibition of tumor growth and viral replication. Currently, numerous drugs become available in American and European markets, and additional 30 antisense nucleic acid drugs are under preclinical study or under phases I, II, and III trial after development.
Due to the large existence of exonucleases in animals, the antisense nucleic acid is quickly degraded and loses the activity if it is not chemically modified. At present, the antisense nucleic acid may be chemically modified through many methods, for example, the common modification of an antisense nucleic acid with phosphorthioate and 2′-methoxy. Moreover, the modification of drugs with phosphorthioate is well studied, and it can effectively resist the degradation by nuclease, and contributes to the activity of the nucleaseRase H. Currently, this modification method is successfully used with the antisense nucleic acid drugs in clinic. However, these are merely modification method for the first generation of antisense nucleic acids. With the development and progression of technologies, new routes and methods of modification will be developed, which allows the research of the second and third generations of antisense nucleic acids to be carried out. Among them, the modification of peptide nucleic acids receives the greatest attention.
Peptide nucleic acids (PNAs) are new analogs of DNA that have neutral amide bonds in the backbone, and can specifically target the groove in DNA. The structural component is N(2-aminoethyl)-glycine, and the bases are attached via methylenecarbonyl to the amino N of the backbone. PNAs are the second generation of antisense nucleic acids.